Propagation and Purification of Chlamydia trachomatis Serovar L2 Transformants and Mutants

Chlamydia trachomatis (C.t.) is an obligate intracellular pathogen that cannot be cultured axenically and must be propagated within eukaryotic host cells. There are at least 15 distinct chlamydial serovariants that belong to 2 major biovars commonly referred to as trachoma and lymphogranuloma venereum (LGV). The invasive chlamydia LGV serovar L2 is the most widely used experimental model for studying C.t. biology and infection and is the only strain with reliable genetic tools available. New techniques to genetically manipulate C.t. L2 have provided opportunities to make mutants using TargeTron and allelic exchange as well as strains overexpressing epitope-tagged proteins, in turn necessitating the regular purification of transformant and mutant clones. Purification of C.t. is a labor-intensive exercise and one of the most common reagents classically used in the purification process, Renografin, is no longer commercially available. A similar formulation of diatrizoate meglumine called Gastrografin is readily available and we as well as others have had great success using this in place of Renografin for chlamydial purifications. Here, we provide a detailed general protocol for infection, propagation, purification, and titering of Chlamydia trachomatis serovar L2 with additional notes specifically pertaining to mutants or recombinant DNA carrying clones.